The science of hematology has long recognized the importance of measuring the amount of hemoglobin in a blood sample since it is the hemoglobin molecule which transports oxygen from the lungs to the various tissues and organs of the body. The accurate measurement of hemoglobin concentration in a patient's blood is arguably the most important parameter determined in a hematology analysis. The hemoglobin concentration is used to screen for anemia which in turn is a sign of underlying disease.
In countries with a "Western Diet", a hemoglobin concentration below 14 grams per deciliter (g/dL) in men and 12 g/dL in women is indicative of anemia. The causes of anemia are many and a low hemoglobin concentration is a strong signal for a thorough workup by the patient's physician. Therefore an accurate and reliable method for measuring hemoglobin concentration is a vital part of any hematology system. The two most common reasons for a patient to be anemic are blood loss and dietary deficiencies in iron, vitamin B12, or Folic Acid. In the former case it is mandatory for the physician to determine the cause of the blood loss and treat it. And again in the latter case, a proper diagnosis is needed to define the appropriate nutritional supplemental treatment.
In addition to its importance as the primary indicator of anemia, the hemoglobin concentration is used in combination with other blood cell parameters to calculate several indices. The Mean Corpuscular Hemoglobin (MCH) is the mass of hemoglobin per red cell and is calculated by dividing the hemoglobin concentration by the number of Red Blood Cells in the comparable volume. The Mean Corpuscular Hemoglobin Concentration (MCHC) is the weight percent of hemoglobin in a Red Blood Cell and is calculated by dividing the hemoglobin concentration by the hematocrit and converting to percent. Both MCH and MCHC are useful parameters in the diagnosis of an anemia. Their importance further emphasizes the necessity of making an accurate and reliable measurement of the hemoglobin concentration in a blood sample.
Modern methods of measuring the hemoglobin content utilize spectrophotometry to quantitate the amount of the oxygen-carrying protein in the sample. The requirements of any spectrophotometric method to measure hemoglobin in a blood sample are two-fold:
1. The method must release all the hemoglobin from the red blood cell in which it is sequestered; and
2. The method must convert all the hemoglobin in the sample into a single chromogenic species, regardless of what form the hemoglobin is in when the reaction is begun.
The first requirement can be achieved by many means; the simplest being to dilute the blood sample in distilled water to effect a hypotonic lysis. However, modern automated hematology instruments require a more rapid lysis than can be achieved with hypotonic lysis. Frequently, surfactants are added to the lysing reagent to hasten the release of hemoglobin and to clear any turbidity which may be in the sample due to elevated lipid content. Various kinds of surfactants are suitable for this task, including anionic, non-ionic, zwitterionic, and cationic. The amount of surfactant required can range from about 100 mg/L to about 50 g/L, depending on the "potency" of the surfactant and the ionic strength of the reagent.
The second requirement necessitates an understanding of the chemistry of the heme iron which carries oxygen when complexed in a globin protein molecule. The heme iron is maintained in the +2 (Fe.sup.II) oxidation state in a normal blood sample. Since the blood sample is usually taken from a vein, the hemoglobin is mostly in the de-oxy state; that is, no oxygen is bound to the heme iron. However, as soon as the sample comes into contact with the atmosphere or is diluted into an oxygen containing buffer or lysing reagent, it is rapidly converted into oxy-hemoglobin; the heme iron binds oxygen but stays in the Fe.sup.II (reduced) state. In many cases, the amount of hemoglobin in the sample could be determined from the oxy-hemoglobin chromogen which is formed naturally upon exposure to air. However, there are some conditions which make this simple solution unacceptable. In some diseases, genetic conditions, or poisonings, a patient may have a significant amount of met hemoglobin in circulation. In met hemoglobin, the heme iron is in the +3, (Fe.sup.III) oxidized state. It cannot bind oxygen, nor can it readily be reduced to Fe.sup.II so that it can bind oxygen to be measured as oxy hemoglobin. Also, heavy cigarette smokers and workers exposed to high concentrations of automobile exhaust frequently accumulate a high concentration of carbon monoxide bound to their heme iron. Carbon monoxide is tightly bound and blocks the binding of oxygen, thereby causing an error in the concentration of hemoglobin if determined by the oxy hemoglobin method. The most commonly used approach to the measurement of hemoglobin is to oxidize all the heme iron to the +3 state and to introduce a ligand which will quantitatively bind to all the heme iron to produce a single chromogenic species for quantitation by spectrophotometry.
The classical method is that of Drabkin. Briefly, the hemoglobin is released by hypotonic lysis (modern adaptations have added surfactants to speed the lysis), the heme iron is oxidized to Fe.sup.III by means of potassium ferricyanide, and the iron reacted with the cyanide anion of potassium cyanide. Cyanide binds very tightly to Fe.sup.III and gives a distinctive chromogen with a peak at about 540 nm. Recent adaptations have involved the deletion of the ferricyanide oxidizing agent and have depended on the oxidation of the heme iron by atmospheric oxygen (or oxygen equilibrated reagents) at elevated pH in the presence of surfactants; cyanide is still used as the heme iron ligand in most procedures in spite of its well-known toxicity.
Many automated hematology analyzers utilize a modification of Drabkin's method. In these methods, red cells are lysed by a cationic surfactant at pH above 10 in the presence of cyanide. Under these conditions, white cell nuclei remain intact and can be counted by common impedance methods. Hemoglobin is measured by taking the optical density of the same solution at 540 nm, as is customarily done with Drabkin's method. The method can yield erroneous measurements in samples which have a high white cell count because of turbidity due to scattering of light by the nuclei. Lipemic and icteric samples also can interfere due to turbidity or increased absorbance of the sample. The present invention avoids these problems by providing a reagent which measures hemoglobin free from interference due to other blood components.
Stroupe, et al. (U.S. Pat. No. 4,200,435) disclose the use of imidazole as a ligand for the determination of glycosylated hemoglobin in the presence of an allosteric effector. They also disclose the use of a surfactant lysing agent, an oxidizing agent, and a heme-binding ligand to determine the amount of hemoglobin in a sample. Stroupe, et at. (U.S. Pat. No. 4,255,385) also disclose reagent kits containing the above reagents. The present invention differs from that disclosed by the Stroupe, et al. patents in that it requires no added oxidizing agent and in that the reaction is completed within 10 seconds as opposed to approximately 10 minutes in the prior art invention.
Benezra, et al. (U.S. Pat. No. 4,853,338) disclose a cyanide-free hemoglobin reagent which comprises a very high surfactant concentration (20 to 50 g/L) and a pH between 11.3 and 13.7. From the elevated pH, and the large amount of N.sub.a OH required to achieve the desired pH, it can be inferred that the heme-binding ligand of this invention is the hydroxide anion. If the hydroxide ion content of the reagent is not high enough, the heme will not be converted stoichiometrically to a single chromogen since water which is present at high (55M) concentration will compete for iron binding sites and may give an erroneous result. The present invention avoids this problem.
It is an object of the present invention to provide a cyanide-free method and reagent for the determination of total hemoglobin present in a whole blood sample. It is a further object of this invention to provide a rapid method for total hemoglobin determination which can be used on automated instruments. It is another object of this invention to provide a cost effective reagent. It is another object of this invention to provide a method for total hemoglobin determination in whole blood without the interference of other blood components.
These and further objects of the invention will become apparent to those of ordinary skill in the art from the following description and figures.